Chemistry Research Journal, 2017, 2(4):118-124

Available online www.chemrj.org

ISSN: 2455-8990
Research Article CODEN(USA): CRJHA5

Preparation, Microstructure and Cytotoxicity Research on Hydroxyapatite/Chitosan-

Chondroitin Sulfate

Qingjiao Han, Mande Qiu*, Yanlong Cui, Meihong Liu

Chemistry and Environmental Science College of Hebei University,Baoding 071002,China

Abstract In this paper, the nano-hydroxyapatite/chitosan-chondroitin sulfate (Hap/Cs-Chs) composite materials

with different mass ratios were prepared by liquid co-precipitation method. On the basis of the microstructure, phase
and compressive strength of the composite materials were investigated respectively. The safety of composite
materials was evaluated by MMT method. The results showed that Hap/Cs-Chs composite materials exhibited
excellent mechanical performance and good biocompatibility. The nano-Hap particles were dispersed uniformly in
the organic phase Cs and Chs with relatively weak crystallinity. It is combined with relatively closely between Hap
particles and Cs-Chs. The particle size is about 40nm-100 nm with spherical shape. The crystallization, particle size,
and dispersion of Hap are influenced directly by the mass ratio of Hap and Cs-Chs. The uniformity, compactness,
and thermal stability can reach the optimum when mass ratio is 50/50. EDS analysis indicated that Ca/P ratio is
around 1.80±0.03, belonging to the calcium-rich type of Hap. The Hela cell toxicity experiment confirmed that the
composite material has good biocompatibility and safety, and the physical and chemical performance can meet the
requirements of bone tissue engineering materials.

Keywords nano-hydroxyapatite, chitosan, chondroitin sulfate, composite materials, microstructure, cytotoxicity

1. Introduction
Hydroxyapatite (Hap) with rich surface properties, good bioactivity, compatibility and bone conductivity, which is
the main inorganic component of human bones and teeth, has been widespread concern in recent years [1-2].
However, the application of Hap in bone tissue engineering has been limited due to its brittleness and low
degradation rate [3-4]. Therefore, it is a hotspot for preparation of functional composite materials satisfying bone
tissue engineering performance and evaluation of various properties of materials for the composite materials [5-6].
According to the present research, in order to overcome the shortcomings of Hap's brittleness and extremely low
degradation rate, it is an effective means to improve the comprehensive performance of materials by the introduction
of other phase material for modification, compound [7-8]. Among them, Hap/Cs composite is considered as the most
promising bone tissue composite because of its good biocompatibility and biodegradability and other advantages [9-
10]. However, the study found that hydroxyapatite/chitosan composite has the problems of low interfacial bonding
between Hap and Cs, uneven dispersion of particles, poor brittleness and strength [11-12]. To overcome this
limitation, more and more researchers introduce other substance for modifying Hap/Cs composite materials.
Chondroitin sulfate (Chs) is a macromolecule classified as glycosaminoglycan found in natural cartilage and other
tissues. Chs has a number of biological properties for tissue engineering including non-toxic, anti-inflammatory,
adhesive, thickening, degradable, promoting cartilage regeneration and good biocompatibility [13-17], and has been
widely used in the biomedical field. As the third phase, it can increase the interface combination between Hap and

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Cs, while giving the composite material excellent mechanical, chemical and biological properties. Undoubtedly, it
has an important theoretical significance and broad prospects. In recent years, many scholars have done a great deal
of research on Hap/Cs-Chs composite materials, most of them mainly focus on their mechanical property, material
preparation. However, there are few studies on microstructure and biological safety evaluation of materials. In this
paper, nano-Hap/Cs-Chs composite materials with different weight ratios were prepared by a liquid co-precipitation
method. Based on the analysis of phase, composition, microstructure and mechanical properties of composites, the
safety of composites was evaluated by MMT method. Which provides a good experimental basis for the practical
application of this series of composite materials.

2. Experimental Section
2.1. Reagent and Instrument
Main reagents: Hela cell line (Institute of Materia Medica, Chinese Academy of Sciences); RPMI-1640 medium
(GIBCOcompany); RPMI-1640 culture medium powder and trypsin were purchased from Gibco company; fetal
bovine serum (FBS) was obtained from Hyclone company, dimethyl sulfoxide was purchased from Sigma company;
MTT was purchased from Amresco company; Chitosan (Cs) with 90 percent degree of the deacetylation,
Chondroitin sulfate were obtained from Weifang Ruiguang Chemical Co., Ltd;. Analytical grade Ca(NO3)2·4H2O,
(NH4)2HPO4, NaOH, acetic acid (HAc).
Main instruments: Y-2000 Automatic X-ray diffraction (Dandong Radiative InstrumentGroup Co. Ltd); JSM-7500F
(Field emission scanning electron microscope) with a Thermo Noran X-ray energydispersive
spectrometer(EDS);Material compressive strength was measured using JWE-50 universal testing machines;
Cytotoxicity tests were performed by BD FACS Calibur flow cytometry (US BD Corporation).

2.2. The Preparation of Compound Materials

In this experiment, cthe solution of Ca(NO3)2•4H2O and (NH4)2HPO4aqueous solution were prepared with near 1.67
Ca/P stoichiometric ratio. The mass ratio of Cs and Chs were fixed at 5:1. Hap/Cs-Chs composite materials with
quality ratio 70/30, 50/50, 40/60 and 30/70 were prepared.
The specific methods are as follows: Cs powder and Chs were dissolved in 2 wt% HAc and deionized water
respectively and mixed completely stirring for 1h. Ca(NO3)2 solution was dispersed in the above solution to form a
homogeneous solution.. Then (NH4)2HPO4solution was dropped into the mixture with vigorous stirring at 40 °C for
6 h, and the pH was adjusted to 10 with NaOH solution. The obtained white slurry was aged for 24 h at room
temperature, and then the precipitate was filtered, washed, and then put them into the mould to prepare composite
samples of different proportions.

2.3. Cytocompatibility evaluation

Hela cells were cultured by adherent culture methods in RPMI1640 cell culture medium with 10% heat-inactivated
fetal bovine serum, 0.2% NaHCO3, and 1% penicilli–streptomycin. The cells were maintained at 37°C under 5%
CO2 with culture medium changed every 2 days. When cell density reached up to 80%, the cells were cultured with
trypsin enzyme-digesting technique and passaged with the ratio of 12. Cells were subcultured every 3 to 4 days.
Cells in logarithmic growth phase were used in MTT assay. The preparation of the material leaching solution was
carried out according to the Biological Evaluation of Medical Devices (ISO10993.5) and related literature [18-21].
The absorbance (OD) value was measured by MTT method at a wavelength of 490 nm under the automatic
microplate spectrophotometer (ELX-800). The survival rate of the blank experimental cells was set at 100% and
used as a reference. The cell viability of the samples under different concentrations of samples (100, 50, 25, 12.5,
6.25mg/mL) was calculated by following formula:
Cell Relative Growth Rate RGR (%) = (Test Group OD / Negative Control Group OD)×100 %
Toxicity of cells was assessed by the cell relative growth rate. When RGR≥80%, the sample showed no toxic effects
on cell proliferation .Otherwise, it has a toxic effect on cell proliferation
3. Results and Discussion

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3.1. XRD Analysis

Figure 1 shows the XRD patterns of Hap/Cs-Chs composite materials with different mass ratio. As can be seen from
Figure 1, the characteristic diffraction peaks of Hap appear at 2θ=26°, 31.8°, 32.3°,32.9°, 39°, which meets with
standard (Ca10(PO4)6(OH)2, PDF file No: 72-1243). The base-line of spectra is high, and the peak shape is wide, no
other crystalline phases could be detected in the XRD patterns. It shows that Hap is a weak crystalline state in the
composite, which is similar to the natural bone [14]. With the content of amorphous Cs-Chs increasing, the relative
intensity of the Hap diffraction peak decreases gradually and the diffraction peak of chitosan appeares at
2θ=20°.This indicates that there is interaction between Hap and Cs Chs. The change of Cs-Chs amount does not
affect the formation of hydroxyapatite phase in the composite material, but it affects the crystallinity and particle
size of Hap.in composite materials. The characteristic diffraction peaks of chondroitin sulfate did not appear in
Figure 1, because Chs is noncrystalline phase and less amount in the composite.

Figure 1: XRD patterns of n-Hap/Cs-Chs composites materialsat different mass ratios

3.2. SEM and EDS analysis

The microstructure and chemical composition of the composites with different mass ratio were observed by SEM
and EDS. As shown in Figure 2(a) and 2(b), the Hap particles can be homogeneously dispersed with Cs and Chs
matrix with good density, and the inorganic phase Hap bonding with organic phase in composite material. There are
a lot of small pores in the composites, which are very helpful for bone cell metabolism of nutrients and moisture
transfer. Figure 2 (c), (d), (e), (f) show the microstructure of different mass ratio of Hap/Cs-Chs composites
materials. The uniform distribution, good crystallinity and near-spherical shape of Hap particles in the composite are
showed by observing SEM. Moreover, the different mass ratio of Hap/Cs-Chs has direct influence on Hap size and
distribution. When the mass ratio of Hap/Cs-Chs is 70/30, the composite particles are relatively loose and the size is
about 40nm. When the mass ratio of Hap/Cs-Chs is 50/50, the particles were featured of good dispersion, high
porosity and the particle size of Hap is about 45nm. It found that microstructure of the composite becomes more
dense as Cs-Chs mass ratio increased, while the porosity and the number of Hap particles decreased. The reasons
may be that during the co-precipitation process, nano hydroxyapatite particles were firstly formed, and Cs-Chs had

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relate to the adhesive effect of the nano hydroxyapatite particles, resulting in the increase of particle size in
microstructure. Especially, the pH value of the compound system was changed instantaneously when the Chs was
added, which affected hydroxyapatite particles formation and crystal growth .The specific reasons and composite
mechanism need further research.
Furthermore, elementary analysis (EDS) results show an average Ca/P atomic ratio of 1.85 and it can be observed
the presence of trace elements Na in composites materials from Figure 2. It belongs to the rich calcium type of Hap
and the existence of sodium is good for natural bone..

Figure 2: SEM photos of composites materials

（a: actual photo; b: cross section; c:70/30; d:50/50; e:40/60; f:30/70）

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Figure 3: EDS spectrum of Hap/Cs-Chs composites materials

3.3 Mechanical Testing

The microstructure of materials is closely related to the macroscopic physical properties. The compressive strength
of the composites was measured using a computer-controlled Universal Testing Machine (JWE-50). Each group
with different mass ratio was tested 3 times, then calculating the average error. The results are shown in table 1. As
can be seen from table 1, the mechanical properties of composite materials is directly affected by the ratio of
Hap/Cs-Chs. When Hap content is 70%, the compressive strength of composite is the smallest (35.2 Mpa). When
Hap/Cs -Chs is 50/50, the compressive strength can be achieved to peak value about 45.3MPa. The toughness of the
composite is improved with a further increase in Cs-Chs, but the compressive strength of the composite decreases
gradually due to the decrease of the inorganic phase. The results are agreement with the results of microstructural
analysis.
Table 1: Compressive strengths of composites materials at different mass ratios
Hap/Cs-Chs Compressive strength (MPa)
70/30 35.2±0.2
50/50 45.3±0.1
40/60 40.6±0.3
30/70 38.2±0.2
3.4. Cytotoxicity tests
Table 2 shows experimental results of Hela cytotoxicity test by MTT method after 24, 48 and 72 hours. As can be
seen from the table, the extract of the composite had no obvious toxic effect on the cells and showed high cell
proliferation rate (RGR>90％). The toxicity graduation of the Hap/Cs-Chs tends to 0 with the extract concentration
decreasing and culture time increasing. According to cell relative growth rate and toxicity grading standards [22-24],
the toxicity rating of the experimental materials ranged from 0 to1, indicating that the Hap/Cs-Chs materials were
not toxic to the Hela cells. The results show that the composite has good cell compatibility and safety to fit the
biological requirements for bone tissue engineering materials.

Table 2: Toxicity experimental results of Hap/Cs-Chs composite materials on Hela cell

Extract Mass Absorbance OD Cell Relative Growth Toxicity Grade
Concentration Rate (RGR %)
(mg/mL) 24h 48h 72h 24h 48h 72h 24h 48h 72h
100 0.3244±0.0014 0.3918±0.0017 0.4805±0.0017 96 95 96 1 1 1
50 0.3245±0.0009 0.396±0.0009 0.4756±0.0012 96 96 95 1 1 1
25 0.3447±0.0014 0.4248±0.0017 0.5406±0.0013 102 103 108 0 0 0
12.5 0.3616±0.0021 0.4537±0.0010 0.5606±0.0016 107 110 112 0 0 0
6.25 0.3684±0.0011 0.4536±0.0021 0.5607±0.0012 109 110 112 0 0 0
Control Hela 0.3380±0.0010 0.4125±0.0008 0.5006±0.0013 100 100 100 0 0 0

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4. Conclusions
 The Nano Hap/Cs-Chs composite materials with different mass ratio were prepared by liquid co-
precipitation method. The noncrystallization structure of hydroxyapatites in the composite material is
similar to that of the natural bone.
 The Hap particle size and dispersion are affected by adding different weight percentages of Cs-Chs, which
exhibits that the particle become more compact with increasing Cs-Chs in composition. The nano-Hap were
dispersed uniformly in the organic phase Cs and Chs with relatively good crystallinity. The particle size of
Hap is about 40-100 nm with a spherical shape. When Hap/Cs-Chs is 50/50, the uniformity, compactness
and compressive strength achieve the best performance.
 Composites containing trace amounts of sodium that is beneficial to the growth of natural bone. The Ca/ P
ratio of composite material is around 1.85, which close to the theoretical value of Hap and belongs to the
rich calcium type of Hap. Cytotoxicity test showed that the Hap/Cs-Chs composite materials had good
biocompatibility and safety. Therefore, the physical and chemical performance of the composite material
can satisfactorily meet the requirements of bone tissue engineering material.

Acknowledgements
This work was financially supported by the Science and Technology Research and Development Projects of the
Hebei Province in China (10276732).

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